Sleeping Beauty(SB)转座子介导增强型绿色荧光蛋白基因(EGFP)体内转染鸡胚胎  

The Enhanced Green Fluorescent Protein Gene(EGFP) Transfer Mediated by Sleeping Beauty Transposon in Chicken(Gallus domesticus) Embryo

作　　者：薛松磊[1] 张立[1] 谢雨琇[1] 李庆平[1] 冯晓军[1] 沈丹[1] 宋成义[1] 高波[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《农业生物技术学报》2013年第2期185-191,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(NO.31200920);江苏省博士后科研资助项目(NO.PD230038);江苏高校优势学科建设工程资助项目(PAPD)

摘　　要：为了探索转座子介导技术在鸡胚中的转染效果,本研究采用鸡蛋开窗法将Sleeping Beauty(SB)单质粒转座载体pT2-SV40-EGFP-SB11注射至孵化36h鸡(Gallus domesticus)胚盘中,同时设开窗不注射组和不开窗组。利用体视荧光显微镜和RT-PCR方法检测增强型绿色荧光蛋白基因(EGFP)表达率,并比较不同阶段的胚胎成活率、孵化率以及蛋重变化。结果表明,EGFP在鸡胚胎中获得较高的表达率(40%～89%);开窗组和不开窗组在胚胎发育早期蛋重变化差异不显著(P>0.05),而后期开窗注射组的蛋重比例显著低于不开窗组(P<0.05);三组胚胎的成活率和孵化率在整个发育过程中变化显著(P<0.05)。本实验初步证明了经改良的鸡蛋开窗法可用于鸡胚转基因研究,并初步建立了SB转座子介导外源基因在鸡胚中转染方法。To explore the effect of transfection of the transposon-mediated technology in the chick embryo, the single plasmid of Sleeping Beauty （SB） transposable vector pT2-SV40-EGFP-SB11 was injected into 36 h incubated chicken （Gallus domesticus） blastoderm with eggs windowing, at the same time window non-injection group and non-window group were set as control group. Stereological fluorescence microscopy and RT-PCR were used to detect and compare the expression of enhanced green fluorescent protein gene （EGFP）, hatching rate, survival rate and egg weight change at different stages of embryonic. The results showed that the transposon-mediated EGFP in chicken embryos obtained a higher expression rate, in embryos of early development （6 d： 89%, 10 d： 71%）, but decreased slightly during later period （14 d： 41%, 18 d： 45%, 21 d： 40%）; The difference of three groups of eggs weight change on 7 d and 12 d ,was not significant （P〉0.05）, the egg weight ratio of the window injection group on 17 d and 21 d, was significantly lower than that of non-window group （P〈0.05）; the difference of survival rate among three groups on 2 d was not significant（P〉 0.05）; window injection group on the 4 d was significantly lower than that of the window non-injection group （P〈0.05）, while the window non-injection group was significantly lower than that of the non-window group（P〈 0.05）; from 6 d to 21 d, the survival rate and hatching rate among the three groups showed significant differences（P〈0.01）. The preliminary experiments proved that the improved egg fenestration can be used for the chick embryo transgenic research and initially establish the method of the transfection exogenous gene in chick embryo mediated with SB transposon.

关 键 词：SB转座子 鸡胚盘 转染 鸡蛋开窗 

分 类 号：S881.2[农业科学—特种经济动物饲养]